Part:BBa_K1321334:Experience

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Applications of BBa_K1321334

This part was cloned into part BBa_K1321333 by Biobrick cloning, to yield BBa_K1321336. The destination vector, containing the AraC-pBAD regulatory elements, was linearized using SpeI and PstI, and AcsAB (previously digested with XbaI and PstI) was ligated at a 1:1 ratio using the T4 ligase. The ligation mix was transformed into chemically competent DH10B Escherichia coli and plated on LB Agar+Chloramphenicol plates, then incubated overnight at 37 degrees. 50 mL Falcon tubes containing 5 ml LB supplied with 50 ug/ml Chloramphenicol were inoculated with a selection of freshly grown colonies for further restriction analysis and gene sequencing verification.

The functionality of part BBa_K1321336 was assayed by inducing the system with 0.1% Arabinose in 5mL LB supplied with 1% Glucose and Chloramphenicol. Overnight incubations were set up at 30 °C and 37 °C, and both empty vector controls and un-induced controls were also assayed for cellulose production. During sonication, LB, soluble and non-soluble fractions were produced and kept for further analysis. Congo Red (CR) at a concentration of 20uM was added to all samples, which were then incubated for 2 hours at room temperature and static conditions, prior to absorbance measurements being taken at 490nm. By subtracting the absorbance values of those samples containing BBa_K1321336 from the absorbance value of a PBS+CR control, it is possible to qualitatively assay the shift in spectral properties of the diazo dye, driven by CR binding to the cellulose fibres.

BBa_K1321335 is made up of two alternative elements of the cellulose synthesis operon, AcsC and AcsD, whose functions are yet not very well known but are believed to play a crucial role in cellulose crystallisation and secretion into the extracellular space. Because these elements were absent, it was expected that the LB and soluble fractions derived from sanitation would contain no cellulose; the first reason being due to the inability of the cells to extrude the fibers, in addition to cellulose being highly hydrophobic hence would tend to precipitate together with the remaining non-soluble, immiscible cellular elements released during sanitation. As expected, no spectral changes were reported in the LB and soluble fractions, however

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